WO2010126272A2

WO2010126272A2 - Method for detecting a ligand using fret biosensor

Info

Publication number: WO2010126272A2
Application number: PCT/KR2010/002632
Authority: WO
Inventors: 이승구; 하재석; 송재준
Original assignee: 한국생명공학연구원
Priority date: 2009-04-27
Filing date: 2010-04-27
Publication date: 2010-11-04
Also published as: EP2426500B1; JP2012525582A; CN102449485B; KR20100117877A; EP2426500A4; JP5710595B2; EP2426500A2; US20120083048A1; US9921215B2; KR101073989B1; WO2010126272A3; CN102449485A

Abstract

The present invention relates to a method for detecting a ligand using a fluorescence resonance energy transfer (FRET) biosensor. More particularly, the present invention relates to a method for simply detecting a ligand within a specimen by measuring the FRET of the biosensor while maintaining a critical temperature, using a phenomenon in which the ligand-binding protein constituting the biosensor is reversibly unfolded at a temperature higher than a specific critical temperature, and the level of such reversible unfolding varies in accordance with the concentration of the ligand. The method of the present invention can be widely applied to various types of FRET biosensors using ligand-binding proteins.

Description

Detection method of ligand using FRET biosensor

The present invention relates to a ligand detection method using a biosensor applying the FRET phenomenon, more specifically, the reversible unfolding of the ligand-binding protein constituting the biosensor appears above a certain critical temperature. In addition, the present invention relates to a method of detecting ligands (especially sugars) at a higher efficiency than the conventional method by using a phenomenon in which the level of structural loosening depends on the concentration of ligands and measuring the concentrations thereof.

In 2002, Frommer at Stanford University developed the first FRET biosensor for maltose measurement (Fehr et al., PNAS., 99: 9846-9851, 2002). Then similar forms of ribose (Lager et al., FEBS Lett., 553: 85, 2003), glucose (Fehr et al., J. Biol. Chem., 278: 19127-19133, 2003), sucrose (Ha et al., Appl. Environ.Microbiol., 73: 7408, 2007) Measurement sensors and the like have been continuously developed.

However, the biosensors developed in the early days have a very low level of detection capability, and therefore, it is necessary to develop a high-sensitivity sensor in order to use a more accurate measuring means (Fehr et al. Current Opinion in Plant Biology, 7: 345, 2004). As part of this effort, the inventors of the Korean Patent Registration 10-0739529 (July 29, 2007) and US Patent US 7432353 (October 7, 2008) disclose a method for optimizing linker peptides between protein domains constituting a biosensor. It is suggested that the detection capability of FRET biosensor for maltose measurement can be increased. In addition, Miyawaki of RIKEN Laboratories significantly increased the detection capability of "Cameleon", a FRET biosensor for calcium measurement by circulating permutation of fluorescent proteins (Nagai et al., PNAS. , 101: 10554, Frommer has increased the detection capability of FRET biosensors by in-frame fusion of glutamine-binding protein (QBP) (Deuschle et al., Protein Sci., 14: 2304, 2006). However, the method of improving the FRET biosensor by genetic manipulation requires a lot of trial and error and a considerable amount of time, and a more efficient and easy improvement method is required since the technical limit has already been reached.

On the other hand, the study to observe the reversible change and thermal stability of the protein structure with the temperature change has been studied to predict the folding process of the protein since the three-dimensional structure of the protein was identified. Interlocked with many researchers, and E. coli-derived periplasmic-binding proteins (PBP) has been a good model for monitoring the thermodynamic changes of these protein structures. According to the observation of the structural change of ARBP (arabinose-binding protein) with temperature using DSC (differential scanning calorimeter), reversible unfolding occurred at 53.5 ℃ in the absence of arabinose. In the presence of 1 mM of arabinose, structural loosening was observed at 59 ° C (Fukuda et al., J. Biol. Chem., 258: 13193. 1983). In addition, in the case of GGBP (glucose / galactose-binding protein), there was a report that the loosening phenomenon with temperature increased from 50 ° C to 63 ° C with or without glucose (Piszczek et al., Biochem. J., 381: 97). , 2004), in the case of MBP, it has been reported that in the presence of maltose, the temperature at which structural annealing occurs increases with pH up to 8-15 ° C (Novokhatny et al., Protein Sci., 6: 141, 1997). .

Therefore, the present inventors have made efforts to improve the ligand concentration measurement and detection ability of the conventional FRET biosensor, and when the ligand is in contact with the biosensor composed of the fusion protein at a specific critical temperature at which a reversible loosening phenomenon occurs. The inventors have found that the ability to detect ligands and measure concentrations has significantly improved and the present invention has been completed.

Summary of the Invention

An object of the present invention is to provide a novel ligand detection and concentration measuring method with improved ligand detection ability and concentration measurement ability of the conventional FRET biosensor.

In order to achieve the above object, the present invention includes a signaling domain including a fluorescence donor and a fluorescence acceptor, and a ligand binding protein connecting the fluorescence donor and the fluorescence receptor. In the ligand detection method using a FRET biosensor including a sensing domain, a reversible unfolding phenomenon occurs and a change in the FRET ratio due to ligand binding is the critical temperature range. It provides a method for detecting a ligand, characterized in that the contact with the sample containing the ligand at a critical temperature.

The present invention also provides a FRET biosensor comprising a signal generator including a fluorescent donor and a fluorescent receptor, and a sensing unit including a ligand binding protein connecting the fluorescent donor and the fluorescent receptor, including the following steps. Provided are methods for measuring ligand concentrations:

(a) contacting the FRET biosensor with a sample containing a ligand at a critical temperature at which a reversible unstructure appears and a change in the FRET ratio as the ligand binds to the ligand binding protein is at the temperature range at which it is greatest; And

(b) measuring the concentration of the ligand by measuring the change in the ratio of the emission amount of the fluorescent donor and the fluorescent acceptor.

Other features and embodiments of the present invention will become more apparent from the following detailed description and the appended claims.

1 is a schematic diagram showing the structural change and FRET efficiency of the FRET biosensor according to the presence or absence of a ligand at room temperature (25 ℃) and critical temperature, resulting in the difference in the amount of light emission of fluorescent proteins.

Figure 2 is a graph showing the change in ratio and Δratio of maltose FRET biosensor by temperature.

3 is a graph showing the change in ratio and Δratio of the glucose FRET biosensor by temperature.

Figure 4 is a graph showing the change in ratio and Δratio of the allose FRET biosensor by temperature.

5 is a graph showing the ratio value and Δratio of the arabinose FRET biosensor with temperature.

FIG. 6 is a titration curve of maltose FRET biosensor measured by ligand concentration at a critical temperature of 25 ° C. and 54 ° C., the maximum temperature of Δratio, and a spectrum of the upper end of the maltose FRET biosensor measured at 25 ° C. and 54 ° C., respectively. Fluorescence spectrum.

7 is a titration curve of a glucose FRET biosensor measured by ligand concentration at a temperature of 25 ° C. and a critical temperature of 50 ° C. having the largest Δratio value, and a spectrum of the upper end of the glucose FRET biosensor measured at 25 ° C. and 50 ° C., respectively. Fluorescence spectrum.

FIG. 8 is a titration curve of an alloose FRET biosensor measured by ligand concentration at a critical temperature of 25 ° C. and a Δratio value of 49 ° C., and the spectrum of the upper part is measured at 25 ° C. and 49 ° C., respectively. The fluorescence spectrum of the sensor.

FIG. 9 is a titration curve of an arabinose FRET biosensor measured by ligand concentration at 25 ° C. and 49 ° C., the critical temperature of which Δratio is the largest. Inset shows fluorescence spectra of arabinose FRET biosensors measured at 25 ° C and 49 ° C, respectively.

10 is a graph illustrating the specificity of the maltose FRET biosensor for various kinds of sugars.

11 is a graph illustrating the specificity of the glucose FRET biosensor for various kinds of sugars.

12 is a graph illustrating the specificity of the allose FRET biosensor for various types of sugars.

FIG. 13 is a graph illustrating the specificity of an arabinose FRET biosensor for various kinds of sugars.

Detailed Description and Specific Embodiments

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

Definitions of main terms used in the detailed description of the present invention are as follows.

As used herein, "fluorescence resonance energy transfer" (FRET) refers to a non-radiative energy transfer phenomenon occurring between two fluorescent materials in different emission wavelengths, and excitation of a fluorescent donor in an excited state. Level energy is transferred to the fluorescence receptor (emission), the emission (emission) is observed from the fluorescence receptor, or fluorescence (quenching) of the fluorescence donor is observed (Lakowicz, JR Principles of Fluorescence Spectroscopy, 2nd ed., New York: Plenum Press, 1999).

As used herein, "fluorescence donor" refers to a fluorescent material that acts as a donor in the FRET phenomenon, and "fluorescence acceptor" refers to a fluorescent material that acts as an acceptor in the FRET phenomenon. Means.

As used herein, the term "ligand-binding protein" refers to a collection of proteins that cause a conformational change by binding a ligand, and includes an E. coli-derived interplasmic binding protein (PBP). (De Wolf et al., Pharmacol Rev., 52: 207, 2000).

As used herein, a "ligand" is a molecule that binds to a ligand binding protein and causes structural changes, such as sugars, amino acids, proteins, lipids, organic acids, metals or metal ions, oxides, hydroxides or conjugates thereof, inorganic It may be any one of ions, amines or polyamines and vitamins, but is not limited thereto.

"Sample" as used herein means a composition that contains or is believed to contain the ligand of interest and will be assayed, and may be any of cells, water, soil, air, food, waste, flora and fauna and flora and fauna. It may be characterized by being collected above, but is not limited thereto. At this time, the flora and fauna includes a human body.

As used herein, the term “critical temperature” refers to a temperature range in which unfolding of the ligand binding protein of the FRET biosensor is controlled by the presence or absence of a ligand to improve detection and measurement capability of the FRET biosensor. That is, the temperature range where the change of FRET ratio according to the binding of ligand to ligand binding protein is greatest. As confirmed in Examples 3 and 4 of the present invention, in the case of the FRET biosensor composed of PBP, a temperature section of 49 to 54 ° C. will be referred to as “critical temperature” in which detection and measurement performance are improved.

In one aspect, the present invention provides a method for detecting a ligand using a FRET biosensor comprising a signal generator including a fluorescent donor and a fluorescent acceptor, and a detector including a ligand binding protein connecting the fluorescent donor and the fluorescent acceptor. In the method for detecting a ligand, the reversible structural loosening occurs, and the ligand is contacted with a sample containing the ligand at a critical temperature at which the change in the FRET ratio as the ligand binds to the ligand binding protein is the greatest. It is about.

Detection of the ligand in the sample is carried out by measuring the amount of luminescence of the fluorescent donor and the fluorescent acceptor with a fluorescence analyzer, etc., a fluorescence spectrometer of a filter method and a monochrome type may be used as the fluorescence analyzer. In this case, when a ligand is present in the sample, a change in the emission amount of the fluorescent donor and the fluorescent acceptor is detected, thereby allowing the ligand to be detected.

In another aspect, the present invention, FRET biosensor comprising a signal generator comprising a fluorescent donor and a fluorescent receptor and a sensing unit comprising a ligand binding protein connecting the fluorescent donor and the fluorescent receptor, including the following steps It relates to a method of measuring ligand concentration using:

(a) contacting the FRET biosensor with a sample containing a ligand at a critical temperature at which a reversible disorganization occurs and the change in FRET ratio as the ligand binds to the ligand binding protein is at a temperature interval where the change is greatest; And

The emission amount of the fluorescent donor and the fluorescent acceptor is measured by a fluorescence spectrometer or the like, and when a change in the concentration of the ligand occurs, a change occurs in the emission amounts of the two fluorescent donors and the fluorescent acceptor. It can be used for measuring concentration change.

In the present invention, the fusion protein constituting the FRET biosensor comprises a fluorescent donor and a fluorescent acceptor as a signal generating portion, and a ligand binding protein as a sensing portion, wherein the fluorescent donor and the fluorescent acceptor are formed of a ligand binding protein. May be bonded at both ends. In this case, the fluorescent donor or the fluorescent acceptor may be linked to the ligand binding protein using one or more linkers.

The ligand binding protein is preferably a maltose-binding protein (MBP), an all-binding protein (ALBP), an arabine-binding protein (ARBP), a galactose / glucose-binding protein (GBP), or the like used in the embodiments of the present invention. It may be characterized in that the E. coli-derived PBP, it is obvious that the ligand binding protein causing a structural change (conformational change) by the binding of the ligand can be provided by the method and sensor according to the present invention.

In addition, the configuration of the fluorescent donor and the fluorescent acceptor used as the signal generator of the biosensor may be any one so long as the emission spectrum of the fluorescent donor and the absorption spectrum of the fluorescent donor overlap each other and cause FRET or fluorescence reduction. As the fluorescent donor, fluorescent proteins, fluorescent pigments, bioluminescent proteins, quantum dots, and the like of various wavelengths may be used as the fluorescent donor, and the fluorescence may be used as the fluorescent acceptor. Fluorescent proteins, fluorescent pigments, quantum dots, etc., which differ in wavelength from the donor, can be used. Alternatively, as fluorescent acceptors, quenchers and Au-nano particles that reduce the fluorescence intensity of the fluorescent donor may be used. However, among the FRET biosensors, ECFP (fluorescent proteins) are considered in consideration of the extinction coefficient, quantum efficiency, photostability and ease of use of the fluorescent donor and the fluorescent acceptor. It is preferable to use enhanced cyan fluorescent protein) and enhanced yellow fluorescent protein (EYFP).

Ligand detection and concentration measurement method according to the present invention uses the optical characteristic of fluorescence "FRET", the principle is shown in FIG. FRET is commonly referred to as resonance energy transfer because the wavelength emitted from the fluorescent donor overlaps with the absorption spectrum of the fluorescent acceptor and occurs without the appearance of photons, which is responsible for the long-range dipole interaction between the fluorescent donor and the fluorescent acceptor. Result. The energy transfer efficiency of FRET is defined by the overlap between the emission spectrum of the fluorescent donor and the absorption spectrum of the fluorescent donor, the quantum efficiency of the fluorescent donor, the relative orientation of the transition dipoles of the fluorescent donor and the fluorescent acceptor, And depends on the distance between the fluorescent donor and the fluorescent acceptor. Therefore, the energy transfer efficiency of FRET is different depending on the distance and relative direction of the fluorescent donor and the fluorescent acceptor. According to Forster's equation, it is expressed as follows.

E = R ₀ ⁶ / (R ⁶ + R ₀ ⁶ ) [Equation 1]

In the above formula, E represents FRET efficiency, and R is a distance between the fluorescent donor and the fluorescent acceptor, which is usually defined to be within 2-9 nm although there are differences depending on the fluorescent material. In addition, R ₀ refers to the distance between the fluorescent donor and the fluorescent acceptor for which the FRET efficiency is 50%, commonly referred to as a Forster distance or Forster radius. R ₀ is represented by the following formula.

R ₀ = 0.211 [ k ² n ^-4 Q _D J (λ)] ^1/6 (in Å) [Equation 2]

In the above formula, k ² is usually calculated as 2/3 as an orientation factor, and has a value ranging from 0 to 4 depending on the relative direction of fluorescence donor emission and fluorescence absorption. n is the refractive index of the medium, and water at 25 ° C. is ˜1.334, and Q _D is the quantum efficiency of the fluorescent donor. J (λ) has a unit value of M ⁻¹ cm ⁻¹ nm ^{4 to} the extent of overlap of the luminescence of the fluorescence donor and the absorption spectrum of the fluorescence acceptor (Lakowicz, JR Principles of Fluorescence Spectroscopy, 2nd ed., New York). Plenum Press, 1999; Patterson et al., Anal. Biochem. 284: 438, 2000; Patterson et al., J. of Cell Sci. 114: 837, 2001).

Therefore, using the above-described principle of FRET, the inventors of the present invention used FRET as a fluorescent donor and a fluorescent acceptor in Korean Patent No. 10-0739529 (July 29, 2007) and US Patent No. 7432353 (October 7, 2008). FRET biosensors were constructed by fusing the fluorescence proteins ECFP (enhanced cyan fluorescent protein) and EYFP (enhanced cyan fluorescent protein) to both ends of ligand-binding protein PBP. It has been shown that quantitatively detects oss, arabinose, ribose, and maltose.

In the FRET biosensor, ECFP-PBP-EYFP is composed of one polypeptide and expressed as a huge fusion protein. The approximate size of PBPs is 3 × 4 × 6.5 nm (Spurlino et al., J. Biol. Chem., 266: 5202, 1991), the distance between the ECFP and the EYFP is approximately 5-6 nm, making it possible to generate FRET. Therefore, when the ECFP is excited at 436 nm, the excitation level energy of the ECFP is transferred to the EYFP so that the emission of the ECFP and the EYFP can be observed simultaneously (see FIG. 1). When the sugar binds to the ligand binding site of the FRET biosensor, the distance and relative direction of ECFP and EYFP fused at both ends of the PBP change, and as a result, a difference in FRET efficiency occurs, so that the ratio of the emission amount of the two fluorescent proteins is increased. Will be different. Therefore, ligands can be detected by measuring the change in the amount of emitted light of two fluorescent proteins. Since the change in the amount of emitted light is proportional to the sugar concentration, quantitative sugar concentration can be measured.

In addition, according to the calculation of Equation 2, ECFP and EYFP have an R ₀ value of approximately 5 nm (Patterson et al., Anal. Biochem., 284: 438, 2000) between ECFP and EYFP. Assuming that the distance of is about 5-6 nm, small changes in distance or relative direction can make a big difference in FRET efficiency. Therefore, the present inventors expected that the detection capability of the biosensor would be greatly improved if the difference in FRET efficiency according to the presence or absence of ligand binding could be maximized. Therefore, in the present invention, as a result of the study to maximize the detection capacity, the EBP-derived PBP shows a reversible structural loosening phenomenon according to the temperature rise, this phenomenon is a study that the structural loosening phenomenon is observed at a higher temperature when the ligand is present In light of the results, a method for detecting and measuring a ligand having increased ligand detection capability was provided in comparison with a method using a conventional FRET principle.

That is, in one embodiment of the present invention it was confirmed that the fluorescence ratio of the biosensor according to the presence or absence of the ligand in the temperature range of 45 ~ 65 ℃ by fluorescence analysis of the FRET biosensors according to the temperature change, more specifically 49 It was confirmed that the "critical temperature" section exists by confirming that the Δratio value, which is the detection capability of the sensor, increases in the temperature range of ℃ ~ 54 ℃ (Fig. In addition, those skilled in the art will understand that the critical temperature of the present invention may be varied in various ranges depending on the stability level and composition of the reaction solution depending on the type of binding protein. That is, biosensors using ligand-binding proteins derived from low-temperature or thermophilic microorganisms or binding proteins with improved substrate specificity are more improved in the temperature range higher or lower than the critical temperature range (49 ° C to 54 ° C) of the present invention. You may have the ability. In particular, substances that affect the structural rigidity of proteins, such as acids, bases, reducing agents, denaturants, chaotropic agents, stabilizers, surfactants, and emulsifiers It is within the ordinary knowledge range expected in the present invention that it is possible to adjust the critical temperature range depending on the presence of an emulsifier or a detergent.

In another embodiment of the present invention, it was confirmed that the detection capacity of the FRET biosensors increased by at least 2.5 to 12 times at the critical temperature than when measured at 25 ℃ (Figs. 6 to 9), the substrate of each sensor Specificity was also confirmed to be improved than the results presented in US Patent No. 7432353 (Fig. 12, Fig. 13).

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Preparation of FRET Biosensor

1-1: Construction of Expression Vector for Preparation of Fusion Protein for FRET Biosensor

First, to provide a biosensor containing a protein represented by the following structural formula I, an expression vector was constructed as follows:

[Formula I]

Wherein the ligand-binding protein PBP is selected from the group consisting of ALBP, ARBP, MBP and GGBP; L ₁ and L ₂ are linker peptides consisting of two amino acids each connecting between the C-terminus of FP ₁ and the N-terminus of PBP, between the C-terminus of PBP and the N-terminus of FP ₂ ; FP ₁ and FP ₂ are FRET fluorescent donors and fluorescent acceptors, which are composed of ECFP and EYFP, respectively.

FRET biosensors capable of quantitatively measuring alloses, arabinose and maltose used in the present invention are sensors disclosed in Korean Patent Nos. 10-0739529 and US Pat. No. 74,32353, previously filed by the present applicants. Same as the above, in detail, it was constructed by the following method.

First, the expression vector of CMY-BII, a maltose biosensor for quantitatively measuring maltose, was constructed by the following method.

SEQ ID NO: 5’-gatcggatccatggtgagcaagggcgag-3 ’

SEQ ID NO: 5'-gatcaagcttgtacagctcgtccatgc-3 '

SEQ ID NO: 5'-gatcatatggtgagcaagggcgag-3 '

SEQ ID NO: 5'-tttaccttcttcgattttcattcgcgacttgtacagctcgtccatgcc-3 '

SEQ ID NO: 5'-atgaaaatcgaagaaggtaaac-3 '

SEQ ID NO: 5'-gatcggatcccgagctcgaattagtctg-3 '

First, the gene of EYFP was a pEYFP-N1 vector (Clontech, Palo Alto, CA) as a template and PCR was performed using primers of SEQ ID NOs: 1 and 2 into which Bam HI and Hind III cleavage sequences were introduced, respectively. The amplified EYFP gene was digested with Bam HI and Hind III restriction enzymes, and inserted into the restriction enzyme recognition site of pET-21a ((Novagen, Madison, WI), which expresses 6 × His-tag at the C terminus of EYFP. PEYFP-III, a possible vector, was constructed by using the pECFP vector (Clontech, Palo Alto, CA) as a template, the sequence number 3 where the restriction enzyme cleavage sequence of NdeI was introduced, and the N-terminal sequence of MBP. PCR was carried out using the overlapping primers of SEQ ID NO: 4. Similarly, the gene of MBP was pMALc2x (NEB, Beverly, MA, USA) as a template, and SEQ ID NO: 5 and Bam HI restriction enzyme cleavage sequence PCR was performed using the primers of SEQ ID NO: 6. Since the genes of each ECFP and MBP thus amplified are overlap-extension PCR by primers produced to overlap each other. SEQ ID NO: 3 and SEQ ID NO: The same amount of ECFP and MBP genes were added to the reaction solution using the primer of No. 6, followed by PCR to obtain an ECFP-MBP type synthetic gene, which was digested with NdeI and BamHI restriction enzymes. The pECMY-BII vector, which is an expression vector, was constructed by cloning the site of the restriction enzyme recognition site of pEYFP-III, the expression vector of MBP-EYFP, and the CMET- FRET biosensor for maltose measurement was constructed as described above. It was named BII.

In addition, amplification of the ECFP gene for constructing the expression vector of CalsBY-QV, a biosensor for detecting alloose, was performed using primers of SEQ ID NOs: 3 and 7. The ALBP gene was a chromosomal DNA extracted from Escherichia coli MG1655. Amplification was carried out using primers SEQ ID NOs: 8 and 9.

SEQ ID NO: 5'-gacagcatattcggcggccattacttgcttgtacagctcgtccatgc-3 '

SEQ ID NO: 5'-atggccgccgaatatgctgt-3 '

SEQ ID NO: 5'-cgcggatcccgattgagtgaccaggatt-3 '

The genes of the amplified ECFP and ALBP were amplified by the synthetic gene of ECFP-ALBP using the primers of SEQ ID NO: 3 and 9. The ECFP-ALBP gene is a pECalsBY-QV vector for the expression of a biosensor for the detection of allose by removing and inserting the ECFP-MBP gene from the pECMY-BII expression vector using Nde I and Bam HI restriction enzyme recognition sites. Was built.

Likewise, the amplification of the ECFP gene for constructing the expression vector of CaraFY-PR, a biosensor for arabinose measurement, was performed using primers of SEQ ID NOs: 3 and 10, and the ARBP gene was based on the chromosomal gene extracted from Escherichia coli MG1655. Amplification was carried out using the primers of SEQ ID NOs: 11 and 12.

SEQ ID NO: 10'-ccgagcttcaggttctccatcctaggcttgtacagctcgtccatgc-3 '

SEQ ID NO: 5'-atggagaacctgaagctcg-3 '

SEQ ID NO: 12 '5-cgcggatcccgacttaccgcctaaacctt-3'

The amplified ECFP and ARBP genes were amplified by a synthetic gene of ECFP-ARBP using primers of SEQ ID NOs: 3 and 12, and pECaraFY-PR was constructed by inserting them at the ECFP-MBP gene position of the pECMY-BII expression vector. .

In addition, the expression vector of CmglBY-SS, a biosensor for glucose measurement, used in the present invention was constructed by the following method.

SEQ ID NO: 13 5′-caccaatgcgagtatcagccatcgaagacttgtacagctcgtccatgcc-3 ’

SEQ ID NO: 14'-atggctgatactcgcattggtg-3 '

SEQ ID NO: 15'-cgcggatcccgatttcttgctgaattcagc-3 '

First, the ECFP gene was subjected to PCR using a pECFP vector as a template, and a reverse primer of SEQ ID NO: 13 prepared by overlapping SEQ ID NO: 3 with the N-terminal sequence of GGBP. Similarly, the GGBP gene was subjected to PCR using a chromosomal gene extracted from Escherichia coli MG1655 as a template and a reverse primer of SEQ ID NO: 14 and SEQ ID NO: 15 to which Bam HI restriction enzyme cleavage sequence was introduced. After adding the ECFP and GGBP genes amplified above to the reaction solution, PCR was performed using primers of SEQ ID NO: 3 and SEQ ID NO: 15 to obtain a synthetic gene of ECFP-GGBP. The amplified ECFP-GGBP synthetic gene was digested with NdeI and BamHI restriction enzymes, and the pECmglBY-SS vector was used to remove and insert the ECFP-MBP gene using the Nde I and Bam HI restriction enzyme recognition sites of the pECMYB-II. Was constructed, and the FRET biosensor for glucose measurement configured as described above was named CmglBY-SS.

1-2: manufacture of FRET biosensor, pure separation

E. coli transformed with pECalsBY-QV, pECaraFY-PR, pECMY-BII and pECmglBY-SS constructed in Example 1-1 to JM109 (DE3), respectively, were treated with LB medium containing 50 μg / ml of ampicillin (1% bacto -trypton, 0.5% yeast extract, 1% NaCl) was incubated for 12 hours at 37 ℃ shaking culture.

Escherichia coli cultured above was inoculated at 1% in 1 L of LB medium to which 50 μg / ml of ampicillin was added and incubated at 37 ° C. for about 2 hours. When the absorbance at 600 nm reached 0.5, IPTG (isopropyl β-d-thiogalactopyranoside) was added to 0.5 mM to induce the expression of proteins at 25 ° C. for 24 hours.

The cultured strains were recovered using a centrifuge (Supra22K, Hanil, Korea) at a speed of 6000 rpm, suspended in 20 mM phosphate buffer (pH 7.5) to destroy the cell membrane with an ultrasonic mill. The dissolved strains were removed again at 15000 rpm by high-speed centrifugation, and the supernatant was filtered through a 0.2 μm filter and used for subsequent purification.

Protein purification was performed using an affinity chromatography column HisTrap ™ HP (GE Healthcare, Uppsala, Sweden) linked to fast-performance liquid chromatography (FPLC) using 6 × His-tag expressed at the C-terminus of FRET biosensors. The first purification was performed, and the second purification was performed using an anion exchange chromatography column HiTrap ™ Q HP (GE Healthcare, Uppsala, Sweden). The purified FRET sensors were used in the examples below, concentrated to a concentration of 10 mg / ml in PBS buffer (pH 7.4) containing 20% glycerol and stored at -70 ℃.

Example 2: Fluorescence analysis method of FRET biosensor

Fluorescence of FRET biosensors was measured using a fluorescence analyzer, Cary Eclipse (Varian Inc., Mulgrave, Australia), under the same conditions that each sensor protein was adjusted to 0.5 μM in 0.5 ml PBS buffer (pH 7.4). The emission spectrum generated by excitation at 436 nm was confirmed by scanning from 450 nm to 600 nm. In addition, as an index indicating FRET efficiency, the ratio of emission intensity at 530 nm of EYFP generated by FRET and ECFP emission at 480 nm was defined as FRET ratio, which is a value substituted in Equation 3 below.

ratio = (530nm / 480nm) [Equation 3]

ratio: The ratio of light emission of EYFP and ECFP.

530 nm: Luminous intensity of EYFP measured by FRET.

480 nm: Emission amount of ECFP measured when the excitation light is 436 nm.

In addition, Δratio, which defines the detection capability of FRET biosensor, was determined according to Equation 4 below.

Δratio = ratio _max -ratio _min [Equation 4]

Δratio: The maximum difference between the ratios of ligands.

ratio _max : The ratio of the ratio measured under the conditions in which the ligand is present.

ratio _min : The ratio of the ratio measured under the absence of ligand.

The titration curves of the ligands of FRET biosensors were S-shaped using the Hill equation, 4-parameter method of Sigmaplot 10.0 (Systat software Inc., USA). was represented by a curve (Sigmoidal curve) of the type, the dissociation constant K _d (dissociation constant) of the ligand for each of the sensor was set at the concentration of ligand Δratio represents a half value in the curve of the S-shape. In addition, the concentration range of ligand that can be measured quantitatively using each sensor was defined as the ligand concentration within the range of 10% to 90% saturated Δratio.

Example 3 Fluorescence Analysis According to Temperature Change of FRET Biosensor

Fluorescence analysis of FRET biosensors at different temperatures was performed by adjusting each sensor to 0.5 μM concentration in 0.5 ml PBS buffer (pH 7.4) and adding FRET under conditions without ligand and 1 mM ligand. The ratio was measured and analyzed. The volume of ligand added to 0.5 ml of FRET biosensor was limited to 5 μl, corresponding to 1/100 of the total volume to prevent excessive dilution of the sensor. The temperature change was measured at intervals of 5 ° C. up to 10 ~ 65 ° C., and at 45 ° C. to 60 ° C., where the ratio was severely changed. All experimental groups performed experiments with the cap closed in a fluorescent cuvette to prevent moisture evaporation. The cuvette reached its target temperature with the thermostat connected to a peltier device. After 3 minutes, the fluorescence was measured.

As a result, as shown in Figures 2 to 5, the critical temperature of the largest change in the FRET ratio according to the presence or absence of ligands of the FRET biosensors purified in Example 1-2, the maltose sensor is 54 ℃ (Fig. 2) Glucose sensor was 50 ℃ (Fig. 3), allose and arabinose sensor was 49 ℃ (Fig. 4, Fig. 5), and there was a slight difference. It was confirmed to be visible.

In addition, as a result of analyzing the titration curves of each FRET biosensor for the ligand at the critical temperature, as shown in Figures 6 to 9, the maltose sensor is 1.22 (Fig. 6), the glucose is 1.6 (Fig. 7), It was confirmed that the alloose sensor exhibits a Δratio value of −0.62 (FIG. 8) and the arabinose sensor of 0.72 (FIG. 9).

When the Δratio values of the FRET biosensors measured above are compared with those measured at 25 ° C., maltose sensor is 7 times (FIG. 6), glucose is 12 times (FIG. 7), and alloose sensor is 3 times (Fig. 8), arabinose sensor is more than 2.5 times (Fig. 9) increased Δratio value, the result is that the detection capability of various types of FRET biosensor can be significantly improved at a certain critical temperature The results directly confirm the claims of the applicants.

The above results can be summarized as shown in Table 1 below.

Table 1

On the other hand, the concentration range of ligand that can be measured by each FRET biosensor was confirmed to be a physiologically significant concentration range, for example, the concentration of glucose of blood, which is important for measuring blood glucose concentration, is 70 to 200 mg / dl. This corresponds to approximately 400-1100 μM. Therefore, by using a glucose sensor for measuring blood glucose concentrations and the blood corresponding to the concentration range of 4 ~ 11 μM, because the addition of a sensor to be 1/100 by volume, the concentration range of the glucose sensor has a K _d value of 9.2 ± 0.5 μM gives the most accurate concentration range.

Example 4. Specificity Analysis for Ligand of FRET Biosensor

Specificity analysis of various ligands of FRET biosensors was performed on 19 kinds of sugars including polysaccharides and sugar alcohols.

The measurement method was adjusted to 0.5 μM concentration of purified FRET biosensors in 0.5 ml PBS buffer (pH 7.4) to add the concentration of each ligand to 100 μM, 1 mM, 10 mM, and the like. Fluorescence was measured after 3 minutes had elapsed from the time point at which the critical temperature obtained in Example 3 was reached.

As a result, as shown in Figure 10 and 12, it was confirmed that the FRET biosensor for measuring maltose and allose do not specifically bind to other types of sugars. In addition, when compared with the results of the US patent US 7432353 previously filed by the present applicants, measured at 25 ℃ it was confirmed that the alloose FRET biosensor is reduced specificity for high concentration of ribose.

Arabinos and glucose FRET sensors, on the other hand, were found to have a very high affinity for galactose as well as previous studies (Vyas et al., J. Biol. Chem. 266, 5226-5237, 1991; Fehr et al., J. Biol. Chem . 278, 19127-19133, 2003), particularly in the case of glucose sensors, were found to have a low level of affinity with many sugars present at high concentrations in addition to galactose (FIG. 11). However, since the sugars present in the blood are present in trace amounts except for glucose, even if the glucose sensor is used for blood glucose measurement, the error caused by other sugars will not be large. According to a recent study, the affinity for galactose is large. Since reduced GGBP has been reported (Sakaguchi-Mikami et al., Biotechnol. Lett. 30: 1453-1460, 2008), there is ample room to improve the specificity of FRET biosensors for glucose measurement.

As described above, the method according to the present invention is a technique based on the phenomenon that the ligand binding protein constituting the biosensor appears to be reversible structure above a certain threshold temperature, and the level of the structure unwinding depends on the ligand concentration. By measuring fluorescence at the critical temperature, the detection capability of the FRET biosensors can be dramatically increased, and it can be widely applied to all kinds of FRET biosensors using ligand-binding proteins and fluorescent proteins. .

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Electronic file attached.

Claims

A signaling domain including a fluorescence donor and a fluorescence acceptor, and a sensing domain including a ligand binding protein connecting the fluorescence donor and the fluorescent acceptor. In the detection method of ligand using FRET biosensor, reversible loosening occurs and contact with sample containing ligand at critical temperature where the change of FRET ratio as the ligand binds to the ligand binding protein is the largest temperature range. Method for detecting a ligand, characterized in that.
A method of measuring ligand concentration using a FRET biosensor comprising a signal generator comprising a fluorescent donor and a fluorescent acceptor, and a sensing unit including a ligand binding protein connecting the fluorescent donor and the fluorescent receptor, including the following steps: :

(a) contacting the FRET biosensor with a sample containing a ligand at a critical temperature at which a reversible disorganization occurs and the change in FRET ratio as the ligand binds to the ligand binding protein is at a temperature interval where the change is greatest; And

(b) measuring the concentration of the ligand by measuring the change in the ratio of the emission amount of the fluorescent donor and the fluorescent acceptor.
The method of claim 1 or 2, wherein the acid, base, reducing agent, denaturant, chaotropic agent, stabilizer, surfactant, adding at least one of a surfactant, an emulsifier, and a detergent, and then contacting the ligand at a changed critical temperature.
The FRET according to claim 1 or 2, wherein the ligand-binding protein comprises a periplasmic-binding protein (PBP) derived from a low temperature or thermophilic microorganism or PBP having improved substrate specificity thereof, in order to change the critical temperature. Contacting the ligand at a changed critical temperature using a biosensor.
The group according to claim 1 or 2, wherein the ligand is composed of sugars, amino acids, proteins, lipids, organic acids, metals or metal ions, oxides, hydroxides or conjugates thereof, inorganic ions, amines or polyamines and vitamins. It is selected from.
The method of claim 1 or 2, wherein the fluorescent donor is selected from the group consisting of a fluorescent protein, a fluorescent pigment, a bioluminescent protein, and a quantum dot; The fluorescent acceptor is selected from the group consisting of a fluorescent protein, a fluorescent pigment and a quantum dot different from the fluorescent donor.
The method of claim 1 or 2, wherein the fluorescent donor is selected from the group consisting of a fluorescent protein, a fluorescent pigment, a bioluminescent protein, and a quantum dot; The fluorescent acceptor is a quencher or Au-nano particles to reduce the fluorescence intensity of the fluorescent donor.
The method of claim 1 or 2, wherein said fluorescent donor or fluorescent acceptor is linked to said ligand binding protein via one or more linkers.
The method of claim 1 or 2, wherein when the ligand binding protein is PBP, the critical temperature is 49 ~ 54 ℃.
The method of claim 1, wherein the critical temperature is about 54 ° C. when the ligand binding protein is a maltose-binding protein (MBP).
The method of claim 1, wherein the critical temperature is about 50 ° C. when the ligand binding protein is GGBP (galactose / glucose binding protein).
3. The method of claim 1, wherein the critical temperature is about 49 ° C. when the ligand binding protein is an allose-binding protein (ALBP) or an arabose-binding protein (ARBP). 4.